The present invention relates to an immunogenic construct, a process for its preparation as well as its use as a vaccine.
The basis for the recognition of foreign immunogens by the immune system is still the subject of intense research. Each individual is exposed time and again to exogenous substances and it is often the case that only a weak immune response is induced against the corresponding substances.
Many vaccines are based on the use of attenuated microorganisms, for example attenuated viruses, which have the disadvantage however that they include virulent material, even if in a weakened form. These can then lead to an infection when using the corresponding attenuated microorganism to immunize humans and animals with an impaired and/or actively suppressed immune system. Disadvantages can also arise by reversion orxe2x80x94in the case of retrovirusesxe2x80x94through recombination.
For these reasons, inactivated microorganisms and/or specially selected, highly purified proteins derived from the corresponding microorganism, polysaccharides or other immunogenic parts of the microorganism are more frequently being administered as vaccines instead of attenuated microorganisms. However, many of the proteins or polysaccharides and/or their epitopes known and used for this are only weakly immunogenic and the corresponding immune response, for example the formation of antibodies, is extremely weak.
Therefore, it is desirable to increase the immune response against weakly immunogenic substances and various methods for this are known in the art.
The use of adjuvants is an example of this. An adjuvant is an auxiliary agent which when administered together or parallel with an antigen increases its immunogenicity and/or influences the quality of the immune response. Hence, the adjuvant can considerably influence the extent of the humoral or cellular immune response for example. Customary adjuvants are aluminum compounds, lipid-containing compounds or inactivated mycobacteria for example. The use of particular carrier substances such as KLH (keyhole Limpet Hemocyanin) is also among the latest current methods to increase immune responses.
Aside from the bacterial products often used for increasing the immunogenicity of weakly immunogenic substances, the use of hepatitis B antigens as a carrier substance is also described in the art.
Hence, an immunogenic hybrid polypeptide consisting of hepatitis B surface antigen (HBsAg) or a fragment thereof which was bound to a further polypeptide component over a native sulfur atom is described in WO 92/11291 for example.
An immunogenic polypeptide conjugate is known from EP 0 271 302 which comprises hepatitis B core antigen (HBcAg) coupled with a further immunogenic polypeptide over an amino acid side-chain group.
A frequently described disadvantage of the hepatitis B antigens used as carrier substances is the immunodominace of these antigens when they are administered together with other immunogens. The immune response against an immunogen bound to hepatitis B antigen is only weakly pronounced by this.
Recombinant flaviviruses are known from WO 93/06214 which contain nucleic acids derived from at least two flaviviruses. These chimeric viruses contain, for example, the region of a nucleic acid which codes for a structural protein of TBEV and is combined with the region of a nucleic acid which codes for a structural protein of a flavivirus differing from TBEV, for example, Dengue virus. These chimeric viruses are described, among others, for use as live vaccines.
An object of the present invention is to provide a new immunogenic construct that enhances the immune response of a weak immunogen and simultaneously avoids the disadvantages known in the art. Additionally, a method for the production of said construct as well as the use as a vaccine are provided according to the invention.
The above problem is solved according to the invention by providing an immunogenic construct comprising as components (i) an inactive flavivirus or a derivative thereof, and (ii) at least one immunogenic component which is bound to the flavivirus and/or to the derivative.
It was surprisingly found that in binding small amounts of an immunogen to a flavivirus and/or a derivative thereof, the immune response against this immunogen is particularly enhanced. This behavior also indicates that an immune response against weak immunogens is generally enhanced through binding to a flavivirus and/or to a derivative thereof.
In the following, the inactive flavivirus is understood as a virus which no longer has the capacity to replicate in a suitable host and is therewith non-infectious.
The flavivirus is preferably an inactivated virus. For example, it can be a yellow fever virus, hepatitis C virus, Dengue virus or a Japanese Encephalitis virus. An inactive or inactivated TBE virus is particularly preferred, a TBE virus of the western subtype (FSME virus) is most preferred.
The flavivirus can be inactivated by a chemical or physical treatment. For example, a chemical treatment of the flavivirus can consist of a treatment with formaldehyde. A physical treatment can be conducted, for example, by heating and/or by a treatment with radiation (UV-irradiation, radioactive irradiation) and/or by ultrasound treatment. It can also be an attenuated virus which has been attenuated, for example, preferably by at least two mutations, by multiple passages in suitable cells or by targeted mutagenesis. The flavivirus can also be a recombinantly produced virus or a sub-viral and/or virus-like particle.
An inactivated whole virus or corresponding flavivirus antigens and/or derivatives of flavivirus are to be understood within the meaning of the invention under the term xe2x80x98inactive flavivirus or a derivative thereofxe2x80x99.
For example, derivatives of a flavivirus can be viral fragments. Within the meaning of the invention, such fragments of a flavivirus are polypeptides, proteins, polysaccharides, nucleic acids or combinations thereof.
For example, the fragment can be a structural protein of a flavivirus or a part of a structural protein. The size of the fragments can strongly vary; a size of 10 kDa can be seen as a minimum size of the fragment.
For example, a derivative of a flavivirus can also be a chemically modified virus fragment, a synthetically produced polypeptide with analogy to a part of the flavivirus or a synthetically produced structure which increases the adjuvant property of the derivative.
The immunogenic component, which is enhanced in its immune response by the inactive flavivirus or a derivative thereof, is a protein, a polypeptide, a polysaccharide or a nucleic acid and/or a combination of two or more of the above mentioned components or an inactive microorganism. In particular embodiments, the protein, polypeptide, polysaccharide or nucleic acid is derived or originates from a virus, bacterium, fungus or parasite or is derived or originates from an allergen.
Should the immunogenic component be derived from a virus, then the virus is preferably selected from the family of the Hepadnaviridae, Herpesviridae, Poxviridae, Adenoviridae, Papovaviridae, Parvoviridae, Retroviridae, Togaviridae or Flaviviridae. For example, the virus can be HIV, herpes simplex virus, influenza virus hepatitis A, B, C, D, G, E or X.
The immunogenic component can be a protein of a virus, for example, gp160, gp120or p24 of HIV, but can also represent a different subunit of the virus, for example, a regulatory protein such as nef or rev of HIV.
The immunogen can be a so-called subunit vaccine, a recombinant vaccine, an inactive whole virus or a virus-like particle.
Should the immunogenic component be derived from a bacterium, this bacterium is preferably selected from the group Bordetella, Haemophilus, Borrelia, Pseudomonas, Corynebacteria, Mycobacteria, Streptococci, Salmonella, Pneumococci, Staphylococci, Clostridia or Helicobacter.
Should the immunogenic component originate from a parasite, then the parasite is preferably selected from the group Amaebida, Trypanosoma or Plasmodium.
Preferably, the immunogenic component differs from the inactive flavivirus or a derivative thereof. In a particularly preferred embodiment, the immunogenic component is selected from a virus group different from flaviviruses.
For formation of the claimed immunogenic construct, the inactive flavivirus or a derivative thereof and the immunogenic component are bound to each other via a covalent bond. In this connection, the claimed immunogenic construct may also be adsorbed to a carrier.
According to a further embodiment, the inactive flavivirus or a derivative thereof and the immunogenic component are bound by adsorption without formation of a covalent bond and are preferably adsorbed to a carrier.
The carrier used relates to materials as they are more closely described in the following.
The invention also comprises methods for the production of the immunogenic construct according to the invention. For example, a preferred method comprises the following steps:
(i) treating the inactive flavivirus or a derivative thereof and/or the immunogenic component with an activator suitable for covalent bonding,
(ii) optionally separating excess activator,
(iii) incubating the treated inactivated flavivirus and/or a derivative thereof and/or the treated immunogenic component, optionally with a non-treated flavivirus and/or derivative thereof, or a non-treated immunogenic component, under conditions which permit the formation of a covalent bond, and
(iv) purifying the construct.
Homo- or preferably hetero-bifunctional cross-linkers are used as the activator for covalent bonding. Such cross-linkers are substances such as N-hydroxysuccinimide esters, imido esters, maleinimido derivatives, N-hydroxysuccinimides, pyridyl disulfides or compounds containing keto groups for example. Excess activator is optionally separated by means of dialysis, centrifugation, filtration, precipitation or with the aid of a chromatographic method.
The construct obtained with the above mentioned steps (i) to (iii) is subjected in the following to a purification. This can be conducted by means of centrifugation, filtration, precipitation, dialysis or a chromatographic method. For example, gel filtration, affinity chromatographic purification or ion exchange chromatography can be employed as chromatographic methods.
The immunogenic construct according to the invention can also be obtained in using a method which comprises the following steps:
(i) incubating the inactivated flavivirus or a derivative thereof and an immunogenic component together with an adsorbing carrier material under conditions which permit the adsorption of the components to the carrier material, and
(ii) separating the construct from the non-adsorbed components.
Pharmaceutically acceptable substances are used as adsorbing carrier materials. Preferably, metals, insoluble or colloidal metal compounds or polymeric compounds and also lipid vesicles are used.
For example, noble metals such as gold or platinum or metals such as aluminum or iron are considered as metals. Insoluble or colloidal metal compounds are, among others, adjuvants such as hydroxides of aluminum, zinc or iron for example.
Polymer compounds are preferably materials as they are used for the production of resorbable and/or biodegradable materials for example. For example, biodegradable microspheres are to be named as such. Additionally, suitable polymer compounds which are not biodegradable but are physiologically accepted and tolerated (for example, latex) can also be employed according to the invention.
Lipid vesicles of various composition and size are also suitable as carrier materials.
In the above alternatives for the production of the construct according to the invention, wherein the inactivated flavivirus and/or a derivative thereof and the immunogenic component are adsorbed to the carrier, the quantitative proportions of inactivated flavivirus to immunogenic component are 200:1 to 1:200.
The construct obtained according to the above described method, which is adsorbed to a carrier material, is subsequently separated, for example, by centrifugation or filtration, and is optionally purified.
The immunogenic construct according to the invention can also be obtained by a genetic engineering method which comprises the following steps:
(i) providing at least one nucleic acid which codes for the flavivirus or a derivative thereof as well as at least one nucleic acid which codes for the immunogenic component,
(ii) inserting the nucleic acids into a suitable expression system,
(iii) expressing the construct encoded by the nucleic acids in suitable host cells,
(iv) purifying the construct, and
(v) optionally producing a bond between the components of the construct.
A nucleic acid and/or a corresponding part of the nucleic acid which codes for a flavivirus and/or a derivative thereof and/or for the desired immunogenic component is used as component (i). The latter codes for an immunogen and/or an epitope which is derived from a virus, bacterium, a fungus or parasite. The virus, bacterium, the fungus or the parasite are selected from the groups already described above.
The construct is expressed by means of a suitable expression system, for example in viral vectors, phages or plasmids which are additionally equipped with regulatory sequences. Expression can occur in eukaryotic as well as prokaryotic cells. Preferably, expression occurs in mammalian cells.
The expressed construct can be purified according to customary methods for protein purification, for example, by means of chromatographic methods.
According to a further embodiment, the immunogenic construct according to the invention can also comprise the following components:
(i) a nucleic acid sequence for an inactivated flavivirus or a derivative thereof with adjuvant function in connection with
(ii) a nucleic acid for an immunogenic component, and
(iii) regulation sequences which ensure the expression of the nucleic acid sequences in a host.
Preferably, the above mentioned immunogenic construct is present as a viral or bacterial expression vector, as a recombinant phage or a naked DNA and/or RNA. Vaccinia virus is particularly preferred as an expression vector. When the construct is present as naked DNA and/or RNA, the corresponding DNA and/or RNA is present in a plasmid which preferably contains a promoter and/or regulatory sequences and can be directly used as such for vaccination.
The invention further encompasses a vaccine which comprises an immunogenic construct according to the invention corresponding to the various alternatives together with a suitable excipient. This vaccine is suitable to induce an immune responds against flavivirus or a derivative thereof and to simultaneously strengthen the immune response against the immunogenic component(s).
Polyvalent vaccines which contain the constructs according to the invention, wherein these constructs have different immunogenic components, are particularly advantageous. The components of the construct according to the invention, namely the inactivated flavivirus or a derivative thereof and the immunogenic component, are present in a weight ratio of 200:1 to 1:200, preferably in a ratio of 80:1 to 20:1. The optimal ratios are dependent on the type of components and must be optimized for each formulation.
The present invention also relates to pharmaceutical preparations which comprise a construct according to the invention and a pharmaceutically acceptable diluent such as an isotonic sodium chloride solution for example. This pharmaceutical preparation is present in a form suitable for parenteral or mucosal administration.
For example, parenteral administration can occur intravenous, intramuscular, subcutaneous or intradermal and the preparation is present for this as a solution, suspension or as a lyophilizate which is to be reconstituted, preferably in a suitable injection syringe. Mucosal administration can occur intranasal, oral, sublingual, intrarectal or intravaginal and the preparation is present for this purpose in a solid form packaged as tablets or in capsules, as a spray or as a suppository for example.
The pharmaceutical preparation can also be enclosed, for example, within liposomes, biodegradable microspheres or virosomes and the release can then occur by different mechanisms, for example, time-released, pulse-released or slow-released.
The invention further relates to a particular immunoglobulin preparation which is obtainable by immunization of a mammal with a construct according to the invention and subsequent isolation of the immunoglobulins from blood, serum, plasma, plasma fractions or mucosal secretions. Immunization for the production of an immunoglobulin preparation according to the invention can occur parenterally or via the mucosa.
Preferably, the immunoglobulin preparation according to the invention essentially contains IgG or IgA. Under the term xe2x80x98essentiallyxe2x80x99, a preparation is understood which contains between 30-100%, preferably 70-100% IgG and/or IgA with respect to the total content of immunoglobulin. Particularly preferred is an immunoglobulin preparation according to the invention which contains more than 90% IgG and/or IgA.
The immunoglobulin preparation according to the invention is preferably subjected to a method for the inactivation of viruses which may be present. Customary methods for the inactivation of viruses are considered for this, for example, treatment with detergents and/or heat treatment or a treatment according to DE 44 34 538.
The present invention also includes the use of an inactive flavivirus for the production of an adjuvant or a carrier.
A kit for the production of the construct according to the invention is also provided by the present invention. This kit contains as a component the inactive, optionally modified, flavivirus or a derivative thereof and the immunogenic component as well as an activator for covalent binding or an adsorbing carrier material.
A kit for the production of the construct according to the invention can also comprise the immunogenic component and the inactive flavivirus or a derivative thereof suitable for binding to the immunogenic component.
A reagent containing the flavivirus and/or a derivative thereof suitable for binding to an immunogenic component is also provided by the present invention. With the aid of this reagent, each desired immunogenic component, preferably a weak immunogen, can be bound to the flavivirus and/or a derivative thereof.
The reagent can be present as a solution, but also as a lyophilizate. By presenting the reagent as a lyophilizate, this is to be suitably reconstituted with water or a water-containing solvent.
The present invention also comprises the use of an inactive flavivirus or a derivative thereof for the production of an adjuvant or a carrier because an immune reaction against a further immunogenic component is enhanced by the inactive flavivirus and/or a derivative thereof. Preferably, the inactive flavivirus and/or a derivative thereof was inactivated according the methods already described above. Preferably, TBE virus, most preferably TBE virus of the western subtype, is used as a flavivirus. The inactive flavivirus or a derivative thereof for use as a adjuvant has the advantage that it is not immunodominant with respect to the immunogenic component with which it is administered, i.e. an immune reaction to the immunogenic component is not suppressed, but instead is promoted.
A further advantage with respect to other adjuvants, for example with respect to Freund""s complete adjuvant (a suspension of mycobacterium in oil), is that no or hardly any side effects arise from its administration.
FIG. 1 serves for closer illustration of the invention. The upper part of FIG. 1 shows the standardization of the gel filtration column (Sephacryl S-500(copyright)) with inactivated FSME virus and/or with tetanus toxoid which serve as the starting materials for the production of the construct according to the invention.
The lower part of FIG. 1 shows the gel filtration analysis of the FSME-tetanus toxoid construct (xe2x80x98constructxe2x80x99) according to the invention in which the tetanus toxoid component is radioactive labeled.
The curve profile shows that, in the case of the conjugate being present, radioactive labeled tetanus toxoid is eluted together with the FSME virus which indicates a bond between the two components.
After treatment of the construct with dithiothreitol (DTT) which leads to cleavage of the components, the radioactivity was found at that place where the uncoupled tetanus toxoid starting material appeared. It can be inferred from this that tetanus toxoid was actually covalently bound via disulfide bridges to the FSME virus before the DTT treatment.
The present invention is more closely illustrated by the following examples without limiting the invention to them.